Methods for treating metabolic disorders using fgf

ABSTRACT

The method provides methods and compositions for treating metabolic disorders such as impaired glucose tolerance, elevated blood glucose, insulin resistance, dyslipidemia, obesity, and fatty liver.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/526,058 filed Oct. 28, 2014, now U.S. Pat. No. 9,072,708, which is adivisional of U.S. patent application Ser. No. 14/184,621 filed Feb. 19,2014, now U.S. Pat. No. 8,906,854, which is a divisional of U.S. patentapplication Ser. No. 13/641,451, filed Jan. 2, 2013, now abandoned,which is the US National Stage of PCT application no. PCT/US2011/032848,filed Apr. 18, 2011, which claims priority to divisional of U.S. Patentapplication Nos. 61/325,255; 61/325,261; and 61/325,253, all filed Apr.16, 2010, the disclosures of each of which are incorporated herein byreference in their entireties.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.DK062434, DK057978, DK090962, DK063491 and HL105278 awarded by TheNational Institutes of Health. The Government has certain rights in theinvention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file fgf1 sequence listing.txt, createdon Jun. 2, 2015, 3,997 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Metabolic disorders such as type 2 diabetes, obesity, and all of therelated complications, are leading causes of mortality. These disordersare associated with the excessive nutritional intake and lack ofexercise of the Western lifestyle, and increasingly that of the rest ofthe world. Type 2 diabetes is a debilitating disease characterized byhigh-circulating blood glucose, insulin, and corticosteroid levels. Theincidence of type 2 diabetes is high and rising and is becoming aleading cause of mortality, morbidity, and healthcare expenditurethroughout the world (Amos et al., Diabetic Med. 14:S1-85, 1997).Diabetes (and insulin resistant conditions) result in elevated levels ofglucose in the blood. Prolonged high blood sugar may cause blood vesseland nerve damage.

Various pharmacological approaches for the treatment of type 2 diabetesare available (Scheen et al., Diabetes Care, 22(9):1568-1577, 1999). Onesuch approach is the use of thiazolidinediones (TZDs), which represent anew class of oral antidiabetic drugs that improve metabolic control inpatients with type 2 diabetes. TZDs (including rosiglitazone (Avandia®)and pioglitazone (Actos®)) command a large share of the currentantidiabetic drug market. TZDs reduce insulin resistance in adipose,muscle, and liver tissues (Oakes et al., Metabolism 46:935-942, (1997);Young et al. Diabetes 44:1087-1092, (1995); Oakes et al., Diabetes43:1203-1210, (1994); Smith et al., Diabetes Obes Metab 2:363-372(2000)). TZDs also lower the levels of free fatty acid (FFA) andtriglycerides.

TZDs administered alone or in combination with metformin haveglucose-lowering effects in patients with type 2 diabetes and reduceplasma insulin concentrations (i.e., in hyperinsulinaemia) (Aronoff etal., Diabetes Care 2000; 23: 1605-1611; Lebovitz et al., J ClinEndocrinol Metab 2001; 86: 280-288; Phillips et al. Diabetes Care 2001;24: 308-315). Abnormalities in lipid levels can also be treated (Day,Diabet Med 1999; 16: 179-192; Ogihara et al. Am J Hypertens 1995; 8:316-320), high blood pressure (Ogihara et al. Am J Hypertens 1995; 8:316-320) and impaired fibrinolysis (Gottschling—et al. Diabetologia2000; 43:377-383). However, there are numerous side effects associatedwith the use of TZDs, such as weight gain, liver toxicity,cardiovascular toxicity, upper respiratory tract infection, headache,back pain, hyperglycemia, fatigue, sinusitis, diarrhea, hypoglycemia,mild to moderate edema, fluid retention, and anemia (Moller, Nature,2001, 414: 821-827). Accordingly, there is a need for improvedtherapeutic approaches to metabolic disorders that have fewer adverseeffects than the available pharmaceutical approaches utilizing TZDs.

BRIEF SUMMARY OF THE INVENTION

Provided herein are compositions and methods for treating a metabolicdisorder in an individual using an FGF-1 compound. Thus, in someembodiments, the invention provides pharmaceutical compositions fortreating a metabolic disorder comprising an FGF-1 compound. In someembodiments, the FGF-1 compound is a functional fragment of FGF-1 (e.g.,amino acids 1-140, 1-141, 14-135, etc.). In some embodiments, the FGF-1compound is a functional analog of FGF-1. In some embodiments, the FGF-1compound is a functional variant of FGF-1. In some embodiments, theFGF-1 compound is an expression vector comprising a sequence encodingthe FGF-1 compound.

In some embodiments, the pharmaceutical composition is formulated forintravenous administration. In some embodiments, the pharmaceuticalcomposition is formulated for subcutaneous or intraperitonealadministration. In some embodiments, the pharmaceutical composition isformulated for a dose of the FGF-1 compound equivalent to 0.01-1 mgFGF-1 per kg body weight of the individual, e.g., equivalent to0.05-0.1, 0.1-0.2, 0.1-0.4, 0.05, 0.1, 0.2, 0.3, 0.4. 0.5 or higher mgFGF-1 per kg body weight. In some embodiments, the composition includesa second therapeutic agent, e.g., a TZD.

In some embodiments, the metabolic disorder is selected from the groupconsisting of elevated blood glucose (e.g., reduced ability to normalizeglucose), impaired glucose tolerance, insulin resistance, type IIdiabetes, obesity, elevated percent body fat, and fatty liver (hepaticsteatosis). In some embodiments, the metabolic disorder is obesity. Insome embodiments, the individual has a BMI of 25 or higher, e.g., 26,28, 30 or greater than 30. In some embodiments, the metabolic disorderis hepatic steatosis. In some embodiments, the metabolic disorder isinsulin resistance. In some embodiments, the metabolic disorder isimpaired glucose tolerance.

In some embodiments, the invention provides methods of making amedicament for use in treating a metabolic disorder comprising an FGF-1compound as described herein. Further provided is use of an FGF-1compound for treating a metabolic disorder in an individual.

Also provided are methods of treating a metabolic disorder in anindividual (treating an individual with a metabolic disorder) comprisingadministering an FGF-1 compound to the individual, thereby treating themetabolic disorder. The metabolic disorder can be selected from thegroup consisting of elevated blood glucose (e.g., reduced ability tonormalize glucose), impaired glucose tolerance, insulin resistance, typeII diabetes, obesity, elevated percent body fat, and fatty liver(hepatic steatosis). In some embodiments, the metabolic disorder isobesity. In some embodiments, the individual has a BMI of 25 or higher,e.g., 26, 28, 30 or greater than 30. In some embodiments, the metabolicdisorder is hepatic steatosis. In some embodiments, the metabolicdisorder is insulin resistance. In some embodiments, the metabolicdisorder is impaired glucose tolerance.

In some embodiments, the FGF-1 compound is a functional fragment ofFGF-1(e.g., amino acids 1-140, 1-141, 14-135, etc.). In someembodiments, the FGF-1 compound is a functional analog of FGF-1. In someembodiments, the FGF-1 compound is a functional variant of FGF-1. Insome embodiments, the FGF-1 compound is an expression vector comprisinga sequence encoding the FGF-1 compound.

In some embodiments, the administering is intravenous. In someembodiments, the administering is subcutaneous or intraperitoneal. Insome embodiments, the dose of the FGF-1 compound administered isequivalent to 0.01-1 mg FGF-1 per kg body weight of the individual,e.g., equivalent to 0.05-0.1, 0.1-0.2, 0.1-0.4, 0.05, 0.1, 0.2, 0.3,0.4. 0.5 or higher mg FGF-1 per kg body weight. In some embodiments, theFGF-1 compound is administered once per day or less, e.g., every secondday, every third day, every week, every other week, or less.

In some embodiments, the method further comprises administering a secondtherapeutic agent to the individual. In some embodiments, the secondtherapeutic agent is administered at the same time (e.g., in the samecomposition) as the FGF-1 compound. In some embodiments, the secondtherapeutic agent is administered at a different time than the FGF-1compound. In some embodiments, the second therapeutic agent is anothertreatment for a metabolic disorder (e.g., a TZD). In some embodiments,the second therapeutic agent targets an associated symptom, e.g., painor high blood pressure.

Further provided are methods of inducing fatty liver in a food animal,e.g., a bird, such as duck or goose. The methods comprise inhibitingFGF-1 in a food animal. In some embodiments, the method comprisesadministering an effective amount of an FGF-1 inhibitor to the foodanimal. In some embodiments, the FGF-1 inhibitor is an antisensecompound specific for FGF-1, e.g., an expression vector comprising asequence encoding the antisense compound. In some embodiments, the FGF-1inhibitor is an antibody (e.g., Shi et al. (2011) IUBMB Life 63:129). Insome embodiments, the FGF-1 inhibitor is an inhibitor of the FGF-1signaling pathway, e.g., a MAP kinase pathway inhibitor such asPD-098059, PD-161570, PD-173074, SU5402, or SB203580. In someembodiments, the FGF-1 inhibitor is administered more than once, e.g.,once/day, or with food. In some embodiments, the FGF-1 inhibitor isadministered in combination with a high fat diet. In some embodiments,the method comprises generating an FGF-1 knockout or genetically alteredFGF-1 inactive food animal, and feeding the animal with a high fat diet.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. FGF-1 gene structure and expression. (A) The expression ofthe FGF-1 gene is directed by three distinct promoters driving theuntranslated exons: 1A, 1B, and 1D (open bars), spaced up to 70 kbapart. Alternative splicing of these untranslated exons to the threecoding exons (closed bars) of the FGF-1 gene results in identical butdifferentially expressed FGF-1 polypeptides. This organization as shownfor human and mouse is evolutionary conserved. Tissue distribution mRNAin mice for (B) FGF-1A, (C) FGF-1B, and (D) FGF-1D.

FIGS. 2A-2F. FGF-1A is a direct transcriptional target of PPARγ.Determination of NR-mediated transcriptional regulation of (A) FGF-1A,(B) FGF-1B, and (C) FGF-1D using luciferase reporter assays. (D)Conserved PPAR response element (PPRE) within the proximal promoter ofFGF-1A relative to the transcription start site (TSS). The sequences areshown from the indicated species, numbered SEQ ID NOs:1-9 from top tobottom. (E) Alignment of the

PPRE within the FGF-1A promoter of different species. Underlineindicates nucleotide variations between the PPREs relative to human.Sequence legend (top to bottom): SEQ ID NOs: 10-17. (F) Species-specificresponse of the FGF-1A promoter to PPARγ using luciferase reporterassays.

FIGS. 3A-3G. Transcriptional regulation of FGF-1A in vivo. Levels ofFGF-1A (A, D) and FGF21 (B, C, E, F) mRNA in WAT and liver of wild-typemice (n=5). (A, B, and C): Fed or overnight fast, with or withoutrosiglitazone (5 mg/kg for 3 days p.o.). (D, E, and F): Fed, 2 weeksHFD, or overnight fast. (G) Levels of FGF-1 protein and variouscomponents of the insulin signaling pathway in WAT of wild-type mice ona normal chow diet vs. 3 months HFD (n=3).

FIGS. 4A-4J. HFD-induced insulin resistance in FGF-1 KO mice. Inresponse to HFD diet, FGF-1 KO mice display (A) normal weight gain, (B)reduced epididymal white adipose (eWAT) weight gain, and (C) increasedliver weight as compared to wild-type littermates (n=6-7). FGF-1 KO micedisplay (D) normal glucose tolerance when fed with control diet, butdevelop HFD-induced insulin resistance as indicated by (E) decreasedglucose tolerance and (F) increased insulin tolerance after 6 mo HFD.(G) histology (H&E) of liver (upper panels) and WAT (lower panels) ofwild-type (left panels) and FGF-1 KO (right panels) animals.Histological analysis of 6-month HFD-treated FGF-1 knockout andwild-type mice. FGF-1 KO mice display (H) normal pancreatic isletmorphology and organization, (I) increased hepatic steatosis, and (J)normal adipocyte size and morphology.

FIG. 5. HFD-induced loss of AKT signaling in WAT of FGF-1 KO mice.Protein levels of FGF-1, AKT, GSK3b, ERK1/2, and actin in liver, BAT,WAT, and muscle of HFD-treated (6 months) FGF-1 KO and wild-type mice(n=3). FGF-1 was not detected in muscle.

FIGS. 6A-6B. FGF-1 and rosiglitazone stimulation of Glut1 expression in3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with FGF-1 (+=50ng/ml, ++=100 ng/ml), rosiglitazone (1 μM), or in combination. (A) mRNA(top) and protein (bottom) levels of Glucose Transporter 1 (Glut1); (B)mRNA (top) and protein (bottom) levels of Glucose Transporter 4 (Glut4).

FIGS. 7A-7H. FGF-1 injection studies in rodents. (A) Fed blood glucosein ob/ob male mice treated with FGF-1 (0.5 mg/kg, s.c.), rosiglitazone(TZD, 5 mg/kg, p.o.), or vehicle. FGF-1 was administered once daily, andblood glucose levels were measured at day 0 (basal levels before FGF-1injection), day 3, and day 6, 1 hour after injection. The values (±SE)shown are the average of the measurements of 5 animals in a group; (B)Sustained glucose lowering effects of FGF-1: Fed blood glucose levels inob/ob mice at indicated time points after the last FGF-1 injection atday 6. (C-H): 72 hrs after the sixth dose, another dose was given andeffects of FGF-1 and TZD on (C) body weight, (D) total body fat, (E)lean weight, (F) weight gain, (G) liver weight, and (H) heart weightwere determined.

FIG. 8. Dose response effect of FGF-1 (s.c., mg/kg) on blood glucoselevels of ob/ob mice. The maximum glucose lowering effects of FGF-1 arereached at 0.5 mg/kg with an EC50 =0.25 mg/kg.

FIG. 9. Effect of FGF-1 (s.c., 0.5 mg/kg) on blood glucose levels ofob/ob mice. The results show that a single s.c. dose reduces bloodglucose for more than 2 days.

FIG. 10. Effect of intravenous FGF1 (0.2 mg/kg) on blood glucose levelsof ob/ob mice. IV administration of FGF1 has acute glucose loweringeffects, which last up to one week.

FIG. 11. Effect of chronic FGF-1 on blood glucose. FGF-1 treatment everythird day results in completely normalized blood glucose in ob/ob mice.

FIG. 12. Effect of chronic FGF1 on food intake. FGF-1 induces a reducedfood intake during the first 1-2 weeks of chronic administration, butafter two weeks food intake returned to normal.

FIG. 13. Effect of chronic FGF-1 on body weight. FGF-1 treatmentresulted in a reduced weight gain during the first week of chronic FGF1administration. After one week, weight gain is similar between controland FGF1-treated mice. This reduced weight gain corresponds with reducedfood intake, but is more durable. Reduced weight gain is evident afterfood intake returns to normal.

FIG. 14. Effect of chronic FGF-1 on total percent body fat. FGF-1treated mice display reduced increase in percent body fat.

FIG. 15. Effect of chronic FGF-1 on percent lean mass. FGF-1 treatedmice display increased lean mass as compared with control mice, furtherindicating that the reduced weight is due to a decrease in the percentbody fat.

FIG. 16. Effect of chronic FGF-1 on glucose tolerance. Untreated miceshow impaired glucose tolerance. After 4 weeks of FGF-1 administration,ob/ob mice display a more rapid and effective capacity to clear glucosefrom the blood, indicating that FGF-1 enhances glucose tolerance.

FIG. 17. Effect of chronic FGF-1 on insulin tolerance. After 4 weeks ofchronic FGF-1 administration, ob/ob mice display increased insulinsensitivity. FGF-1 treated mice clear glucose from the blood moreeffectively than untreated mice.

FIG. 18. Effect of chronic FGF-1 on serum lipids. Serum levels oftriglycerides, free fatty acids, and cholesterol are similar betweencontrol and FGF-1 treated mice.

FIGS. 19A and 19B. Effect of chronic FGF-1 on hepatic steatosis. H&Estaining of liver of A) control and B) FGF1-treated ob/ob mice. Controlmice show mixed micro and macro vesicular steatosis with some periportalsparing. Steatosis affects most hepatocytes (>70%). There is little ifany inflammatory infiltrate in either the portal tracts or lobules,which is typical liver histology for an ob/ob mouse. In contrast, liversfrom FGF-1 treated mice display clearing of fat in a periportal to midzonal distribution. Steatosis is dramatically reduced compared tocontrol and is mainly microvesicular. There is very littlemacrovesicular steatosis, and little or no inflammation.

FIG. 20. Effect of chronic FGF-1 on hepatic glycogen. FGF-1 treated icedisplay increased levels of hepatic glycogen as compared to controlmice.

FIG. 21. PPARγ binds to the FGF-1 promoter region in mature adipocytes.Chromatin was prepared from differentiated 3T3-L1 adipocytes andchromatin immunoprecipitation assays were performed with either IgGantibodies (negative control) or anti-PPARγ antibodies. Quantitative PCRdemonstrates that PPARγ specifically binds the FGF1 promoter region.36b4 is a negative control locus devoid of PPARγ binding sites.

FIG. 22. Assessment of delivery method on FGF-1 blood glucose effects.Subcutaneous, intraperitoneal, and intravenous delivery of FGF-1 (0.5mg/kg) display similar efficacy in normalizing blood glucose levels ofob/ob diabetic mice.

FIG. 23. Assessment of delivery method on duration of FGF-1 activity.Single subcutaneous (sc) or intravenous (iv) injection of FGF-1 (0.5mg/kg) in ob/ob mice. FGF-1 glucose normalizing effects persist longerwhen administered iv as compared to sc.

FIG. 24. FGF-1 effects in db/db mice. Single subcutaneous injection ofFGF-1 (0.5 mg/kg) normalizes blood glucose in db/db leptin receptormutant diabetic mice. The db/db model is considered to represent a lesssevere diabetes model than ob/ob. The results indicate that FGF-1 iseffective for treatment of less severe metabolic disorders.

FIG. 25. FGF1 effects in DIO mice. Single subcutaneous injection of FGF1(0.5 mg/kg) normalizes blood glucose in diet-induced obesity mice(C57BL/6). Again, the results indicate that FGF-1 is effective fortreatment of metabolic disorders arising from a number of causes.

FIG. 26. Human recombinant FGF-1 is effective in mice. Singlesubcutaneous injection of human FGF1 (0.5 mg/kg) normalizes bloodglucose in ob/ob mice.

FIG. 27. Comparison of FGF1, FGF2, FGF9, and FGF10 effects. Singlesubcutaneous injection of FGFs (0.5 mg/kg) in ob/ob mice. Only FGF1 hasglucose normalizing effects.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Provided herein are methods and compositions useful for treatingmetabolic disorders using FGF-1 and functional variants thereof. Theinventors have shown that FGF-1 has rapid and long-lasting effects,including normalizing blood glucose, increasing insulin sensitivity,reducing percent body fat and overall body weight, increasing percentlean mass, and reducing fatty liver (hepatic steatosis).

II. Definitions

The following abbreviations are used herein:

FGF fibroblast growth factor

NHR nuclear hormone receptor

PPAR peroxisome proliferator-activated receptor

PPRE PPAR response element

TSS transcription start site

TZD thiazolidinedione

BAT brown adipose tissue

WAT white adipose tissue

HFD high fat diet

i.p. intraperitoneal injection

s.c. subcutaneous injection

p.o. oral administration

i.v. intravenous injection

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

The term FGF-1 compound refers to FGF-1 or a variant thereof (FGF-1fragment, FGF-1 portion, modified form of FGF-1, protein havingsubstantial identity to FGF-1, FGF-1 analog, etc.) that retains at leastone FGF-1 activity (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80% or higher percent activity compared to FGF-1). Thus, FGF-1 compoundsinclude functional FGF-1 fragments, functional FGF-1 variants, andfunctional FGF-1 analogs. An example of an FGF-1 compound that issubstantially identical to FGF-1 is a protein having at least 80%, 85%,90%, 95%, 98%, 99%, or 100% amino acid identity to FGF-1. In someembodiments, the FGF-1 compound comprises a polypeptide having, e.g.,95%, 98%, 99% or higher % identity to FGF-1, where the non-identitiesrepresent conservative substitutions or additions or deletions that donot substantially change the activity.

FGF-1 (or acidic FGF) is a secreted protein that binds heparin (e.g.,heparin sulfate) and FGF receptor family members 1 and 4. The humanprotein is 155 amino acids in length, and the sequence is publicallyavailable at SwissProt accession number P05230.1. The term “FGF-1”refers to naturally-occurring, isolated, recombinant, orsynthetically-produced proteins. FGF-1 also includes allelic variantsand species homologs.

FGF-1 activities include binding heparin, FGFR1, and FGFR4, andincreasing expression of GLUT1 and/or GLUT4. FGF-1 activities alsoinclude (among others) reducing glucose levels, improving glucosetolerance, and increasing insulin sensitivity in a diabetic individual.Additional FGF-1 activities include reducing percent body fat, fattyliver disease, and increasing percent lean mass in an individual.

A functional FGF-1 fragment is a protein having less than the fulllength sequence of FGF-1 but retaining at least 25, 50, or 80% activityof at least one FGF-1 activity (e.g., FGF-1 (14-135, 1-140, 13-135,1-141, etc.). The functional FGF-1 fragment can have an amino acidsequence of any length up to the full length FGF polypeptide sequence,e.g., 50, 50-80, 50-100, 120-150, 100-150, or more than 100 amino acids.In some embodiments, the functional FGF fragment is at least 80%, 85%,90%, 95%, 98%, or 100% identical to FGF-1 over the covered portion ofthe full length sequence (e.g., over 50-150 amino acids). In someembodiments, the functional FGF-1 fragment has greater than 90%, e.g.,95%, 98%, 99% or higher % identity to FGF-1 1-141. In some embodiments,the functional FGF-1 fragment has greater than 90%, e.g., 95%, 98%, 99%or higher % identity to FGF-1 1-141, where the non-identities representconservative substitutions or additions or deletions that do notsubstantially change the activity.

A functional FGF-1 analog is a modified or synthetic (e.g.,peptidomimetic) form of FGF-1 that retains at least 25, 50, or 80%activity of at least one FGF-1 activity. Examples of FGF-1 analogs thatretain heparin-binding activity are disclosed in WO2006/093814. TheFGF-1 analog can include non-naturally occurring amino acids, ormodified amino acids, e.g., that improve the stability (in storage or invivo) or pharmacological properties (tissue profile, half-life, etc.) ofthe protein. The functional FGF-1 analog can also be a functional FGF-1variant, e.g., having greater than 90%, e.g., 95%, 98%, 99% or higher %identity to FGF-1. In some embodiments, the functional FGF-1 analog hasat least 95%, 98%, 99% or higher % identity to FGF-1, where thenon-identities represent conservative substitutions or additions ordeletions that do not substantially change the activity.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA.

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing.

The words “protein”, “peptide”, and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers, those containingmodified residues, and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following amino acids aretypically conservative substitutions for one another: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids, or two or more polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides, or amino acids, that are the same (i.e.,about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection. See e.g.,the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are thensaid to be “substantially identical.” This definition also refers to, ormay be applied to, the compliment of a nucleotide test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions. Algorithms can account forgaps and the like. Identity generally exists over a region that is atleast about 25 amino acids or nucleotides in length, or over a regionthat is 50-100 amino acids or nucleotides in length.

The term “metabolic disorder” is used broadly herein to refer to theconditions, diseases, and disorders associated with insulin and/orglucose dysregulation. Metabolic disorders include type 2 diabetes,insulin insensitivity, glucose intolerance, elevated blood glucoselevels, obesity, high percent body fat, fatty liver, etc. One of skillwill understand that metabolic disorders are associated with and canresult in a wide range of other disorders, e.g., high blood pressure,heart disease, poor circulation, etc., which can be ameliorated byaddressing the metabolic disorder according to the methods of theinvention.

“Biopsy” or “biological sample from a patient” as used herein refers toa sample obtained from a patient having, or suspected of having, ametabolic disorder. In some embodiments, the biopsy is a blood sample,which can be separated into blood components (plasma, serum, white bloodcells, red blood cells, platelets, etc.). In some embodiments, thesample is a tissue biopsy, such as needle biopsy, fine needle biopsy,surgical biopsy, etc. Tissue samples can be obtained from adipose,muscle, liver, etc.

A “biological sample” or “cellular sample” can be obtained from apatient, e.g., a biopsy, from an animal, such as an animal model, orfrom cultured cells, e.g., a cell line or cells removed from a patientand grown in culture for observation. Biological samples include tissuesand bodily fluids, e.g., blood, blood fractions, lymph, saliva, urine,feces, etc.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as livestock and companionanimals. The term does not necessarily indicate that the subject hasbeen diagnosed with a metabolic disorder, but typically refers to anindividual under medical supervision. A patient can be an individualthat is seeking treatment, monitoring, adjustment or modification of anexisting therapeutic regimen, etc. The terms can refer to an individualthat has been diagnosed, is currently following a therapeutic regimen,or is at risk of developing a metabolic disorder, e.g., due to familyhistory, sedentary lifestyle, etc.

A “control” condition or sample refers to a sample that serves as areference, usually a known reference, for comparison to a test conditionor sample. For example, a test sample can represent a patient sample,while a control can represent a sample from an individual known to havea metabolic disorder, or from an individual that is known to not havethe disorder. In another example, a test sample can be taken from a testcondition, e.g., in the presence of a test compound, and compared tosamples from known conditions, e.g., in the absence of the test compound(negative control), or in the presence of a known compound (positivecontrol). A control can also represent an average value gathered from anumber of tests or results. One of skill in the art will recognize thatcontrols can be designed for assessment of any number of parameters. Forexample, a control can be devised to compare therapeutic benefit basedon pharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of benefit and/or side effects). One of skill in the art willunderstand which controls are valuable in a given situation and be ableto analyze data based on comparisons to control values. Controls arealso valuable for determining the significance of data. For example, ifvalues for a given parameter are widely variant in controls, variationin test samples will not be considered as significant.

The terms “therapy,” “treatment,” and “amelioration” refer to anyreduction in the severity of symptoms. In the case of treating metabolicdisorders, the terms can refer to reducing blood glucose, increasinginsulin sensitivity, reducing body weight, reducing percent body fat,increasing percent lean mass, reducing side effects of associatedtherapies, etc. As used herein, the terms “treat” and “prevent” are notintended to be absolute terms. Treatment can refer to any delay inonset, amelioration of symptoms, improvement in patient survival,increase in survival time or rate, etc. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment, or to the same patient prior to treatment or at a differenttime during treatment. In some aspects, the severity of disease isreduced by at least 10%, as compared, e.g., to the individual beforeadministration or to a control individual not undergoing treatment. Insome aspects the severity of disease is reduced by at least 25%, 50%,75%, 80%, or 90%, or in some cases, no longer detectable using standarddiagnostic techniques.

The terms “effective amount,” “effective dose,” “therapeuticallyeffective amount,” etc.

refer to that amount of the therapeutic agent sufficient to ameliorate adisorder, as described above. For example, for the given parameter, atherapeutically effective amount will show an increase or decrease oftherapeutic effect at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%,80%, 90%, or at least 100%. Therapeutic efficacy can also be expressedas “-fold” increase or decrease. For example, a therapeuticallyeffective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold,or more effect over a control. In the context of the present invention,the effective amount of an FGF-1 compound can vary depending onco-administration of other therapeutics or metabolic profile of theindividual (among other factors such as age, severity of disease, etc.).

The term “diagnosis” refers to a relative probability a subject has agiven metabolic disorder. Symptoms and diagnostic criteria aresummarized below. Similarly, the term “prognosis” refers to a relativeprobability that a certain future outcome may occur in the subject. Forexample, in the context of the present invention, prognosis can refer tothe likelihood that an individual will develop a metabolic disorder.Prognosis can also refer to the likely severity of the disease (e.g.,severity of symptoms, rate of functional decline, survival, etc.). Theterms are not intended to be absolute, as will be appreciated by any oneof skill in the field of medical diagnostics.

III. Fibroblast Growth Factor (FGF)-1

Fibroblast growth factors (FGFs) are a family of distinct polypeptidehormones that are widely expressed in developing and adult tissues(Baird et al., Cancer Cells, 3:239-243, 1991). FGFs play crucial rolesin multiple physiological functions including angiogenesis, development,mitogenesis, pattern formation, cellular proliferation, cellulardifferentiation, metabolic regulation, and repair of tissue injury(McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998;Beenken and Mohammadi, 2009). The FGF family now consists of at leasttwenty-three members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res.313:139-157 (2003).

FGFs bind to FGF receptors (FGFRs), of which there are four (FGFR1-4).The receptor binding specificity of each FGF is distinct, and can alsodepend on the particular isoform of the FGFR. For example FGFR1 has atleast 3 isoforms that result in different splice variants in the thirdIg-like domain (Lui et al. (2007) Cancer Res. 67:2712). FGF signaling isalso determined by the tissue specificity of the receptor and receptorisoform. FGF-1 can bind to all FGFRs, but is reported to be internalizedonly upon binding to FGFR1 and FGFR4. A review of FGF-FGFR specificitiescan be found, e.g., in Sorensen et al. (2006) J Cell Science 119:4332.

The polypeptide and coding sequences of FGF-1 are known for a number ofanimals and publically available from the NCBI website. FGF-1 compoundsthat can be used in the methods of the invention include full lengthhuman FGF-1, species homologs thereof, and functional fragments thereof.Additional FGF-1 compounds that can be used include modified versions ofFGF-1 (e.g., modified to increase stability, e.g., PEGylated orincluding non-naturally occurring amino acids), functional analogs ofFGF-1, and functional FGF-1 variants with substantial identity to FGF-1.Another FGF-1 compound that can be used in the present methods includesexpression vectors for stable or transient expression of FGF-1 in acell. FGF-1 compounds include those that retain at least one FGF-1activity, e.g., binding heparin, FGFR1, and FGFR4, and increasingexpression of GLUT1 and/or GLUT4. FGF-1 activities include (amongothers) reducing (normalizing) glucose levels, improving glucosetolerance, and increasing insulin sensitivity in a diabetic individual.Additional FGF-1 activities include reducing percent body fat, fattyliver disease, and increasing percent lean mass in an individual.

In some embodiments, the FGF-1 compound is a functional FGF-1 variant,functional FGF-1 fragment, and/or functional FGF-1 analog. That is, theFGF-1 compound can be a functional FGF-1 fragment with variations andmodified or non-naturally occurring amino acids, as long as the FGF-1compound retains at least one FGF-1 activity. In some embodiments, theFGF-1 compound is substantially identical to full length FGF-1 or afragment thereof, e.g., at least 95, 98, or 99% identical over therelevant length of FGF-1, where the non-identities include conservativesubstitutions or deletions or additions that do not affect the FGF-1activity. Examples of FGF-1 amino acids that are involved in FGF-1activities, and thus less amenable to substitution or deletion, includeTyr-15, Arg-35, Asn-92, Tyr-94, Lys-101, His-102, Trp-107, Leu-133, andLeu-135. Also included are Lys-112, Lys-113, Lys-118, Arg-122, andLys-128, which are involved in heparin interactions. The position ofthese residues is with reference to the 140 amino acid human sequence ofmature FGF1, but can be determined for species homologs.

In some embodiments, the FGF-1 compound comprises amino acids 1-140 ofFGF-1, or a sequence having at least 90% identity to amino acids 1-140of FGF-1 that retains at least one FGF-1 activity. In some embodiments,the FGF-1 activity is normalizing blood glucose levels in an individual.In some embodiments, the FGF-1 activity is reducing percent body fat inan individual. In some embodiments, the FGF-1 activity is increasinginsulin sensitivity in an individual. In some embodiments, the FGF-1activity is binding to FGFR1 or FGFR4. In some embodiments, the FGF-1activity is increasing expression of GLUT1.

The FGF-1 compound may be generated, isolated, and/or purified by anymeans known in the art. For standard recombinant methods, see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY (1989); Deutscher, Methods in Enzymology 182: 83-9(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, NY (1982).

The FGF-1 compound can be modified, e.g., to improve stability or itspharmacological profile. Chemical modifications include, e.g., addingchemical moieties, creating new bonds, and removing chemical moieties.Modifications at amino acid side groups include acylation of lysineε-amino groups, N-alkylation of arginine, histidine, or lysine,alkylation of glutamic or aspartic carboxylic acid groups, anddeamidation of glutamine or asparagine. Modifications of the terminalamino group include the des-amino, N-lower alkyl, N-di-lower alkyl, andN-acyl modifications. Modifications of the terminal carboxyl groupinclude the amide, lower alkyl amide, dialkyl amide, and lower alkylester modifications.

Examples of compounds that can improve the pharmacological profile ofthe FGF-1 compound include water soluble polymers, such as PEG, PEGderivatives, polyalkylene glycol (PAG), polysialic acid, hydroxyethylstarch, peptides (e.g., Tat (from HIV), Ant (from the Drosophilaantennapedia homeotic protein), or poly-Arg), and small molecules (e.g.,lipophilic compounds such as cholesterol or DAG).

In some embodiments, the FGF-1 is linked to a heparin molecule, whichcan improve the stability of FGF-1, and prevent interaction with heparinin vivo. Linking heparin to FGF-1 ensures that more of the modifiedFGF-1 remains in circulation than it would without the heparinmodification.

The FGF-1 compound can be expressed recombinantly using routinetechniques in the field of recombinant genetics. Standard techniques areused for cloning, DNA and RNA isolation, amplification and purification.Generally enzymatic reactions involving DNA ligase, DNA polymerase,restriction endonucleases and the like are performed according to themanufacturer's specifications. Basic texts disclosing the generalmethods of use in this invention include Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the seriesAusubel et al. eds. (2007 with updated through 2010) Current Protocolsin Molecular Biology, among others known in the art.

To obtain high level expression of a nucleic acid sequence, such as thenucleic acid sequences encoding an FGF-1 compound, one typicallysubclones a nucleic acid sequence that encodes a polypeptide sequence ofthe invention into an expression vector that is subsequently transfectedinto a suitable host cell. The expression vector typically contains astrong promoter or a promoter/enhancer to direct transcription, atranscription/translation terminator, and for a nucleic acid encoding aprotein, a ribosome binding site for translational initiation. Thepromoter is operably linked to the nucleic acid sequence encoding apolypeptide of the invention or a subsequence thereof.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto the recombinant polypeptides to provide convenient methods ofisolation, e.g., His tags. In some case, enzymatic cleavage sequences(e.g., Met-(His)g-Ile-Glu-GLy-Arg which form the Factor Xa cleavagesite) are added to the recombinant polypeptides. Bacterial expressionsystems for expressing the polypeptides are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available.

Standard transfection methods can be used to produce cell lines thatexpress large quantities of polypeptides of the invention, which arethen purified using standard techniques (see, e.g., Colley et al., J.Biol. Chem., 264:17619-17622 (1989); Guide to Protein Purification, inMethods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformationof cells is performed according to standard techniques (see, e.g.,Morrison, J. Bact., 132:349-351 (1977); Clark-Curtiss & Curtiss, Methodsin Enzymology, 101:347-362 (Wu et al., eds, 1983). For example, any ofthe well known procedures for introducing foreign nucleotide sequencesinto host cells may be used. These include the use of calcium phosphatetransfection, polybrene, protoplast fusion, electroporation, liposomes,microinjection, plasma vectors, and viral vectors (see, e.g., Sambrooket al., supra).

FGF-1 can be purified to substantial purity by standard techniques knownin the art, including, for example, extraction and purification frominclusion bodies, size differential filtration, solubility fractionation(i.e., selective precipitation with such substances as ammoniumsulfate); column chromatography, immunopurification methods, etc.

The FGF-1 compound can also be chemically synthesized using knownmethods including, e.g., solid phase synthesis (see, e.g., Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963) and Abelson et al., Methods inEnzymology, Volume 289: Solid-Phase Peptide Synthesis (1st ed. 1997)).Polypeptide synthesis can be performed using manual techniques or byautomation. Automated synthesis can be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer).Alternatively, various fragments of the polypeptide (and any modifiedamino acids) can be chemically synthesized separately and then combinedusing chemical methods to produce the full length polypeptide. Thesequence and mass of the polypeptides can be verified by GC massspectroscopy. Once synthesized, the polypeptides can be modified, forexample, by N-terminal acetyl- and C-terminal amide-groups as describedabove. Synthesized polypeptides can be further isolated by HPLC to apurity of at least about 80%, preferably 90%, and more preferably 95%.

The invention further provides methods of inhibiting FGF-1 to inducefatty liver in a food animal, e.g., a bird such as a duck, goose, quail,etc. The inhibited expression or activity can be 40%, 50%, 60%, 70%,80%, 90% or less than that in a untreated or wild type control. Incertain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, or more in comparison to a control.

Typically, inhibition of FGF-1 is accompanied by a high fat diet. Insome cases, the method comprises generating a genetically modifiedanimal with defective FGF-1 activity (e.g., an FGF-1 knockout animal).In some embodiments, FGF-1 is inhibited by administering an FGF-1inhibitor to the animal. Typically, the inhibitor is administered morethan once, e.g., on a regular schedule (daily, weekly, etc.) or withfood.

The FGF-1 inhibitor can be an antisense compound. The term “antisense”is used herein as a general term referring to RNA targeting strategiesfor reducing gene expression. Antisense includes RNAi, siRNA, shRNA,etc. Typically, the antisense sequence is identical to the targetedsequence (or a fragment thereof), but this is not necessary foreffective reduction of expression. For example, the antisense sequencecan have 85, 90, 95, 98, or 99% identity to the complement of a targetRNA or fragment thereof. The targeted fragment can be about 10, 20, 30,40, 50, 10-50, 20-40, 20-100, 40-200 or more nucleotides in length.

The term “RNAi” refers to RNA interference strategies of reducingexpression of a targeted gene. RNAi technique employs genetic constructswithin which sense and anti-sense sequences are placed in regionsflanking an intron sequence in proper splicing orientation with donorand acceptor splicing sites. Alternatively, spacer sequences of variouslengths can be employed to separate self-complementary regions ofsequence in the construct. During processing of the gene constructtranscript, intron sequences are spliced-out, allowing sense andanti-sense sequences, as well as splice junction sequences, to bindforming double-stranded RNA. Select ribonucleases then bind to andcleave the double-stranded RNA, thereby initiating the cascade of eventsleading to degradation of specific mRNA gene sequences, and silencingspecific genes. The phenomenon of RNA interference is described anddiscussed in Bass, Nature 411: 428-29 (2001); Elbahir et al., Nature411: 494-98 (2001); and Fire et al., Nature 391: 806-11 (1998); and WO01/75164, where methods of making interfering RNA also are discussed.

The term “siRNA” refers to small interfering RNAs, that are capable ofcausing interference with gene expression and can causepost-transcriptional silencing of specific genes in cells, for example,mammalian cells (including human cells) and in the body, for example, ina mammal (including humans). The siRNAs based upon the sequences andnucleic acids encoding the gene products disclosed herein typically havefewer than 100 base pairs and can be, e.g., about 30 bps or shorter, andcan be made by approaches known in the art, including the use ofcomplementary DNA strands or synthetic approaches. The siRNAs arecapable of causing interference and can cause post-transcriptionalsilencing of specific genes in cells, for example, mammalian cells(including human cells) and in the body, for example, in a mammal(including humans). Exemplary siRNAs have up to 40 bps, 35 bps, 29 bps,25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integerthereabout or therebetween. Tools for designing optimal inhibitorysiRNAs include that available from DNAengine Inc. (Seattle, Wash.) andAmbion, Inc. (Austin, Tex.).

A “short hairpin RNA” or “small hairpin RNA” is a ribonucleotidesequence forming a hairpin turn which can be used to silence geneexpression. After processing by cellular factors the short hairpin RNAinteracts with a complementary RNA thereby interfering with theexpression of the complementary RNA.

The FGF-1 inhibitor can also be an antibody that interferes with FGF-1signaling, e.g., a FGF-1 specific antibody, or a functional fragmentthereof. An example of an FGF-1 antibody is described, e.g., in Shi etal. (2011) IUBMB Life 63:129, but several are commercially available.Antibodies can exist as intact immunoglobulins or as any of a number ofwell-characterized fragments that include specific antigen-bindingactivity. Typically, the “variable region” of the antibody contains theantigen-binding activity, and is most critical in specificity andaffinity of binding. See Paul, Fundamental Immunology (2003). Suchfragments can be produced by digestion with various peptidases. Pepsindigests an antibody below the disulfide linkages in the hinge region toproduce F(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region, therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially Fab with part of the hinge region (see FundamentalImmunology (Paul ed., 3d ed. 1993). While various antibody fragments aredefined in terms of the digestion of an intact antibody, one of skillwill appreciate that such fragments may be synthesized de novo eitherchemically or by using recombinant DNA methodology. The term antibodyincludes antibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990)).

The FGF-1 inhibitor can also be an inhibitor of the FGF-1 signalingpathway, e.g., a MAP kinase pathway inhibitor such as PD-098059,PD-161570, SU5402, or SB203580.

IV. Metabolic Disorders Amenable to Treatment with an FGF-1 Compound

The FGF-1 compound described herein can be used to treat metabolicdisorders, e.g., type 2 diabetes, insulin insensitivity, glucoseintolerance, metabolic syndrome, fatty liver disease, obesity, andconditions related thereto. Related to the obesity application, theFGF-1 compound can also be used to reduce percentage body fat and/orincrease the percentage of lean mass in an individual. Conditionsrelated to the metabolic disorders, that can also benefit from treatmentwith and FGF-1 compound include high blood pressure (hypertension),cardiovascular disease, hyperglycemia, hyperuricemia, and polycysticovary syndrome.

Metabolic syndrome (also known as metabolic syndrome X or syndrome X) isa combination of medical disorders that increases the risk ofcardiovascular disease. In general, a diagnosis of metabolic syndromerequires at least three of the following criteria (see InternationalDiabetes Foundation (IDF) and U.S. National Cholesterol EducationProgram (NCEP)):

Central obesity: waist circumference≧40 inches (male), ≧36 inches(female)

BMI: >30 kg/m²

Elevated triglycerides (dyslipidemia): >150 mg/dL

Lowered HDL cholesterol: <40 mg/dL (males), <50 mg/dL (females)

Raised blood pressure (BP) (hypertension): systolic BP>130 or diastolicBP>85 mm Hg

Raised fasting plasma glucose (FPG): >100 mg/dL

Elevated LDL cholesterol is marked by levels above about 100, about 130,about 160 or about 200 mg/dL. Metabolic syndrome may also be related toelevated total cholesterol.

Impaired glucose intolerance is defined as a two-hour glucose levels(glycemia) of about 140 to about 199 mg/dL (7.8 to 11.0 mmol) on the75-g oral glucose tolerance test (according to WHO and ADA). Glycemia ofabout 200 mg/dl or greater is considered diabetes mellitus.

Hyperglycemia, or high blood sugar, can be defined as a blood glucoselevel higher than about 7, about 10, about 15, or about 20 mmol/L.

Hypoglycemia, or low blood sugar, can be defined as preprandial bloodglucose below about 4 or about 6 mmol/L (72 to 108 mg/dl) or 2-hourpostprandial blood glucose below about 5 or about 8 mmol/L (90 to 144mg/dl).

Insulin resistance is defined as a state in which a normal amount ofinsulin produces a subnormal biologic response. Insulin resistance canbe measured by the hyperinsulinemic euglycemic clamp technique,Homeostatic Model Assessment (HOMA), or Quantitative insulin sensitivitycheck index (QUICKI).

Hyperuricemia is an abnormally high level of uric acid in the blood,e.g., above 360 μmol/L (6 mg/dL) for women and 400 μmol/L (6.8 mg/dL)for men.

Polycystic ovarian syndrome (PCOS) is associated with oligoovulation,anovulation, excess androgen, and/or polycystic ovaries. Metabolicsyndrome may also be associated with acanthosis nigricans.

Metabolic syndrome may also be associated with a pro-inflammatory state(e.g., elevated C-reactive protein levels in the blood, e.g., above 10mg/L) and microalbuminuria (urinary albumin excretion ratio≧20 mg/min oralbumin:creatinine ratio≧30 mg/g).

In some embodiments, the FGF-1 compound can be used to treat fatty liverdisease or a condition related thereto. The fatty liver disease can be amethod of treating nonalcoholic steatohepatitis (NASH), nonalcoholicfatty liver disease (NAFLD), simple fatty liver (steatosis), cirrhosis,hepatitis, liver fibrosis, or steatonecrosis. Fatty liver disease can beassessed by diagnostic methods known in the art including liver enzymetests (ALT, AST), liver ultrasound, FibroTest®, SteatoTest®, coagulationstudies including international normalized ratio (INR), as well as bloodtests including M30-Apoptosense ELISA, erythrocyte sedimentation rate,glucose, albumin, and renal function.

Fatty liver disease may also be associated with a pro-inflammatory state(e.g., elevated C-reactive protein levels in the blood, e.g., above 10mg/L) as well as hepatocellular carcinoma. Fatty liver disease may alsobe associated with abetalipoproteinemia, glycogen storage diseases,Weber-Christian disease, Wolman disease, acute fatty liver of pregnancy,lipodystrophy, inflammatory bowel disease, HIV, and hepatitis C(especially genotype 3), and alpha 1-antitrypsin deficiency.

In some embodiments, the FGF-1 compound is used to reduce percentagebody fat, increase percentage lean mass, or to treat obesity (as well asassociated conditions). The method can be used to treat class I obesity,class II obesity, class III obesity, elevated body weight, elevated bodymass index (BMI), elevated body volume index (BVI), elevated body fatpercentage, elevated fat to muscle ratio, elevated waist circumference,or elevated waist-hip ratio.

Class I obesity is characterized by a BMI of about 30 to about 35, classII obesity (severe obesity) is characterized by a BMI of about 35 toabout 40, and class III obesity (morbid obesity) is characterized by aBMI of 40 or greater. A BMI of greater than about 45 or 50 is consideredsuper obese. Elevated body weight can be assessed in consideration ofage, gender, height, frame, and/or ethnicity.

Elevated waist-hip ratio is defined as greater than about 0.9 for menand greater than about 0.7 for women.

Metabolic disorders are inter-related and can result in disorders acrossvarious systems. Addressing the core metabolic disorder can reduce theseverity of related conditions in a patient, including, e.g.:

cardiovascular disorders including, e.g., ischemic heart disease, anginaand myocardial infarction, congestive heart failure, high bloodpressure, abnormal cholesterol levels, deep vein thrombosis, andpulmonary embolism,

neurological disorders including, e.g., stroke, meralgia paresthetica,migraines, idiopathic, and intracranial hypertension,

depression (especially in women) and social stigmatism,

rheumatological and orthopedic disorders including, e.g., gout, poormobility, osteoarthritis, and lower back pain,

dermatological disorders including, e.g., stretch marks, acanthosisnigricans, lymphedema, cellulitis,

gastrointestinal disorders including, e.g., gastroesophageal refluxdisease (GERD) and cholelithiasis (gallstones),

respiratory disorders including, e.g., obstructive sleep apnea, obesityhypoventilation syndrome, asthma, and increased complications duringgeneral anaesthesia,

urology and nephrology disorders including, e.g., erectile dysfunction,urinary incontinence, chronic renal failure, and hypogonadism.

V. Pharmaceutical Compositions

The FGF-1 compounds can be used and formulated into any of a number ofpharmaceutical compositions, including those described in the UnitedStates Pharmacopeia (U.S.P.), Goodman and Gilman's The PharmacologicalBasis of Therapeutics, 10^(th) Ed., McGraw Hill, 2001; Katzung, Ed.,Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange, 8^(th)ed., Sep. 21, 2000; Physician's Desk Reference (Thomson Publishing;and/or The Merck Manual of Diagnosis and Therapy, 18^(th) ed., 2006,Beers and Berkow, Eds., Merck Publishing Group; or, in the case ofanimals, The Merck Veterinary Manual, 9^(th) ed., Kahn Ed., MerckPublishing Group, 2005.

The compositions disclosed herein can be administered by any means knownin the art. For example, compositions may include administration to asubject intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intrathecally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularly, orally, locally, by inhalation, byinjection, by infusion, by continuous infusion, by localized perfusion,via a catheter, via a lavage, in a cream, or in a lipid composition.Administration can be local, e.g., to adipose tissue or to the liver, orsystemic.

Solutions of the active compounds as free base or pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered and the liquid diluent firstrendered isotonic with sufficient saline or glucose. Aqueous solutions,in particular, sterile aqueous media, are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage can be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion.

Sterile injectable solutions can be prepared by incorporating the activecompounds or constructs in the required amount in the appropriatesolvent followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium.Vacuum-drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredients, can be usedto prepare sterile powders for reconstitution of sterile injectablesolutions. The preparation of more, or highly, concentrated solutionsfor direct injection is also contemplated. DMSO can be used as solventfor extremely rapid penetration, delivering high concentrations of theactive agents to a small area.

Heparin can interfere with FGF-1 circulation when the FGF-1 compound isnot administered intravenously. For non-i.v. administration, e.g.,subcutaneous administration, the FGF-1 compound can be linked to aheparin molecule, or another compound that interferes with FGF-1 bindingto heparin. The FGF-1-heparin interaction in vivo reduces the amount ofcirculating FGF-1, and the duration of the therapeutic effect. Thus, insome embodiments, the invention provides a pharmaceutical compositioncomprising an FGF-1 compound linked to heparin. Diabetes medications arecommonly administered s.c., thus, it can be more convenient to thepatient to receive the FGF-1 compound in the same s.c. composition, orin a different composition but using a familiar route of administration.

Pharmaceutical compositions can be delivered via intranasal or inhalablesolutions or sprays, aerosols or inhalants. Nasal solutions can beaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions can be prepared so that they aresimilar in many respects to nasal secretions. Thus, the aqueous nasalsolutions usually are isotonic and slightly buffered to maintain a pH of5.5 to 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, and appropriate drug stabilizers, ifrequired, may be included in the formulation.

Oral formulations can include excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. In someembodiments, oral pharmaceutical compositions will comprise an inertdiluent or assimilable edible carrier, or they may be enclosed in hardor soft shell gelatin capsule, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 75% of the weight of the unit, or preferably between25-60%. The amount of active compounds in such compositions is such thata suitable dosage can be obtained.

In some embodiments, FGF-1 is administered using a gene therapyconstruct, e.g., as described in Nikol et al. (2008) Mol Ther. Thus, insome embodiments, an individual is treated for a metabolic disorder byadministering to the individual an expression vector comprising asequence that codes for a FGF-1 compound. Similarly, the methods ofinducing fatty liver in an animal can rely on administration of anexpression vector, in this case, an expression vector encoding anantisense construct specific for FGF-1.

In some cases, a polynucleotide encoding FGF-1 is introduced into a cellin vitro and the cell is subsequently introduced into a subject. In somecases, the cells are first isolated from the subject and thenre-introduced into the subject after the polynucleotide is introduced.In some embodiments, FGF-1-encoding polynucleotides or FGF-1 inhibitorypolynucleotides are introduced directly into cells in the subject invivo.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding FGF-1 polypeptides in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding FGF-1 polypeptides, or FGF-1 inhibitory polynucleotidesto cells in vitro. In some embodiments, such polynucleotides areadministered for in vivo or ex vivo gene therapy uses. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome. Viral vectordelivery systems include DNA and RNA viruses, which have either episomalor integrated genomes after delivery to the cell. For a review of genetherapy procedures, see Anderson, Science 256:808-813 (1992); Nabel &Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166(1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460(1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne,Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids encoding engineeredpolypeptides of the invention include lipofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described, e.g., in U.S.Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No.4,897,355, and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, WO 91/17424, WO 91/16024.Delivery can be to cells (ex vivo administration) or target tissues (invivo administration). The preparation of lipid:nucleic acid complexes,including targeted liposomes such as immunolipid complexes, is wellknown to one of skill in the art (see, e.g., Crystal, Science270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995);Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al.,Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722(1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos.4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728,4,774,085, 4,837,028, and 4,946,787).

RNA or DNA viral based systems can be used to target the delivery ofpolynucleotides carried by the virus to specific cells in the body anddeliver the polynucleotides to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used totransfect cells in vitro. In some cases, the transfected cells areadministered to patients (ex vivo). Conventional viral based systems forthe delivery of polypeptides of the invention could include retroviral,lentivirus, adenoviral, adeno-associated and herpes simplex virusvectors for gene transfer. Viral vectors are currently the mostefficient and versatile method of gene transfer in target cells andtissues. Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene, and hightransduction efficiencies.

VI. Methods of Treatment

The invention provides methods of treating, preventing, and/orameliorating a metabolic disorder in a subject in need thereof. Thecourse of treatment is best determined on an individual basis dependingon the particular characteristics of the subject. The treatment can beadministered to the subject on a daily, twice daily, every other day,bi-weekly, weekly, monthly or any applicable basis that istherapeutically effective. The treatment can be administered alone or incombination with at least one other therapeutic agent, e.g., targetingthe same metabolic disorder or a related symptom. The additional agentcan be administered simultaneously with the FGF-1 compound, at adifferent time, or on an entirely different therapeutic schedule (e.g.,the FGF-1 compound can be administered daily, while the additional agentis weekly).

The suitability of a particular route of administration will depend inpart on the pharmaceutical composition, its components, and the disorderbeing treated. Parenteral administration is often effective for systemictreatment.

The dosage of a therapeutic agent administered to a patient will varydepending on a wide range of factors. For example, it would be necessaryto provide substantially larger doses to humans than to smaller animals.The dosage will depend upon the size, age, sex, weight, medical historyand condition of the patient, use of other therapies, the potency of thesubstance being administered, and the frequency of administration.

The dose of the FGF-1 compound can be equivalent to 0.005-1 mg FGF-1 perkg body weight. For example, the dose can be equivalent to 0.01-0.1,0.1-0.2, 0.1-0.5. 0.2-0.5, 0.5-0.8. or 0.5 or more mg FGF-1 per kg bodyweight. One of skill will understand and be able to adjust to situationswhere the FGF-1 compound is smaller (e.g., a functional FGF-1 fragment)or larger (e.g., a modified FGF-1 polypeptide) than FGF-1.

Having indicated that there is variability in terms of dosing, it isbelieved that those skilled in the art can determine appropriate dosingby administering relatively small amounts and monitoring the patient fortherapeutic effect. If necessary, incremental increases in the dose canbe made until the desired results are obtained. Generally, treatment isinitiated with smaller dosages which may be less than the optimum doseof the therapeutic agent. Thereafter, the dosage is increased by smallincrements until the optimum effect under circumstances is reached. Thetotal daily dosage can be divided and administered in portions duringthe day if desired.

The pharmaceutical preparation can be packaged or prepared in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g.,according to the dose of the therapeutic agent. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation. The composition can, if desired, also contain othercompatible therapeutic agents.

In some embodiments, the FGF-1 compound is co-administered with at leastone additional therapeutic agent, e.g., another therapeutic agent fortreating a metabolic disorder, or a therapeutic agent to addressassociated symptoms, e.g., a blood thinner or analgesic. Therapeuticagents commonly used for metabolic disorders include drugs from thefollowing classes: alpha-glucosidase inhibitors, amylin agonists,dipeptidyl-peptidase 4 (DPP-4) inhibitors, meglitinides, sulfonylureasand PPAR agonists such as thiazolidinediones (TZD). The PPAR agonist,e.g., PPARγ agonist, can include, e.g., aleglitazar, farglitazar,muraglitazar, tesaglitazar, and thiazolidinedione (TZD). Exemplary TZDsinclude pioglitazone (Actos®), rosiglitazone (Avandia®), rivoglitazone,and troglitazone (Hauner, Diabetes Metab Res Rev 18:S10-S15 (2002)).

Additional complementary active agents, such as biguanides (e.g.,metformin) or sulfonylureas, can also be used in appropriatecircumstances.

The combination of an FGF-1 compound with another therapeutic agent canresult in a synergistic effect with enhanced efficacy in the treatmentof metabolic disorders such as type 2 diabetes and related conditions.The synergy allows for reduced dosages of the active agents incombination as compared to the dosages for either active individually.The reduced dosage can help reduce any side effects that may appear.

Accordingly, in combination therapy, the effective amount of theadditional (second) therapeutic agent and the effective amount of theFGF-1 compound are together effective to reduce the symptoms/effects ofmetabolic disorder. In some embodiments, the combination is an FGF-1compound and TZD. The FGF/TZD combination allows for a reduced dose ofTZD required for therapeutic treatment of type 2 diabetes, therebyminimizing the side effects typically observed with TZD therapy. Forexample the amount of TZD administered in combination with the FGF-1compound is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or toabout 80% compared to the typical dose of TZD utilized in the treatmentof type 2 diabetes.

One of skill in medicine can best determine the appropriate dose of theadditional therapeutic agent by considering the state of the patient,the recommended dose, the severity of disease, and the synergisticeffect of the FGF compound. For example, the amount of rosiglitazone canbe about 4 mg to about 8 mg per day (e.g., about 2 mg, about 4 mg, orabout 8 mg per dose). The amount of pioglitazone can be about 15 mg toabout 45 mg per day, e.g., about 30 mg per day.

The following discussion of the invention is for the purposes ofillustration and description, and is not intended to limit the inventionto the form or forms disclosed herein. Although the description of theinvention has included description of one or more embodiments andcertain variations and modifications, other variations and modificationsare within the scope of the invention, e.g., as may be within the skilland knowledge of those in the art, after understanding the presentdisclosure. All publications, patents, patent applications, Genbanknumbers, and websites cited herein are hereby incorporated by referencein their entireties for all purposes.

VII. EXAMPLES Materials and Methods

Animals. The animals that were used were FGF-1^(-/-) (Miller et al.,2000), PPARγ^(f/f)/aP2-Cre mice (He et al., 2003) and wild-typelittermate controls of a >99% C57/B6 genetic background.

Ob/ob male mice (8 wks old, B6.Cg-Lep^(ob)Ldlr^(tm1Her)/J) were fromJackson labs. The Ob/ob mouse model is an animal model forhyperglycemia, insulin resistance, and obesity. Male ob/ob mice are usedto monitor plasma glucose levels, lipid levels, etc.

Animals were kept in a temperature-controlled environment with a 12-hourlight/12-hour dark cycle. They received a standard diet (MI laboratoryrodent diet 5001, Harlan Teklad) or high fat (60%), high carbohydrate(HFD) diet (F3282, Bio-Serv), and acidified water ad libitum.

Cell culture. 3T3-L1 mouse pre-adipocytes were from American TypeCulture Collection (ATCC, Rockville, Md.). Cells were maintained atsub-confluence in growth medium (GM) containing 10% calf serum inDulbecco's modified Eagle's medium (DMEM) at 37° C. and 5% CO₂. Forstandard adipocyte differentiation, cells were stimulated at 2 days postconfluency (referred as day 0) with differentiation medium (DM)containing 10% fetal bovine serum (FBS), 5 μg/ml insulin, 1 μMdexamethasone, and 0.5 μM 3-isobutyl-1-methylxanthine (IBMX) for 48hours. Then, the medium was replaced with DMEM 10% FBS with 5 μg/mlinsulin for an additional 48 hours. Then, the cells were maintained inpost differentiation medium containing 10% FBS. CV-1 cells were used forluciferase reporter assays. CV-1 cells were cultured in DMEM medium with10% fetal bovine serum at 37° C. and 5% CO₂.

Western analysis. Total cell lysates from tissues were prepared asdescribed. Western blotting was performed as described using polyclonalgoat anti-human FGF-1 (C-19) antibody (1:200, Santa Cruz), anti-AKT(1:1000, Cell Signaling Technology, 9272), monoclonal rabbit anti-GSK3b(1:1000, Cell Signaling Technology, 9315), and polyclonal rabbitanti-p44/42 MAPK (1:1000, Cell Signaling Technology, 9102). Antibodybinding was detected using peroxidase-conjugated donkey anti-goat IgG(1:5000, Santa Cruz).

Serum analysis. Blood was collected by tail bleeding either in the adlibitum fed state or following overnight fasting. Free fatty acids(Wako), triglycerides (Thermo), and cholesterol (Thermo) were measuredusing enzymatic colorimetric methods following the manufacturer'sinstructions. Serum insulin levels were measured using an UltraSensitive Insulin ELISA kit (Crystal Chem). Serum adiponectin levelswere measured by ELISA (Millipore). Plasma adipokine levels weremeasured using a Milliplex™ MAP kit (Millipore).

Histological analysis and immunohistochemistry. Tissues were fixed in 4%phosphate-buffered formalin, embedded in paraffin, sectioned at 4 μm,and stained with hematoxylin and eosin according to standard procedures.For immunohistochemistry, tissues were deparaffinized in xylene andrehydrated. Slides were incubated with 5% normal donkey serum in PBS(+0.2% Triton-X100 and 1% BSA) for 30 min, and subsequently sectionswere incubated overnight with a 1:200 dilution of primary antibodies at4° C. and using Alexa Fluor 488 or 595 as secondary antibodies for 2 hrsat RT.

Metabolic studies. Glucose tolerance tests (GTT) were conducted afterovernight fasting. Mice were injected intraperitoneally (i.p.) with 1 gof glucose per/kg body weight, and blood glucose was monitored at 0, 15,30, 60, 90, and 120 min using a OneTouch Ultra glucometer (LifescanInc). Insulin tolerance tests (ITT) were conducted after overnightfasting. Mice were injected i.p. with 0.5 U of insulin/kg body weight(Humulin R; Eli Lilly), and blood glucose was monitored at 0, 15, 30,60, 90, and 120 min using a OneTouch Ultra glucometer (Lifescan Inc).Real-time metabolic analyses were conducted in an undisturbed room under12 h/12 h light/dark cycles using a Comprehensive Lab Animal MonitoringSystem (Columbus Instruments).

In Example 5, ob/ob male mice (8 wk old) were randomized into threegroups and treated with daily subcutaneous (s.c.) injections ofrecombinant mouse FGF-1 (0.5 mg/kg in PBS), oral rosiglitazone (TZD, 5mg/kg in 0.5% carboxymethyl cellulose), or vehicle. Blood glucose levelswere measure in fed animals one hour after treatment. Total bodycomposition analysis was performed using an EchoMRI-100™ (Echo MedicalSystems, LLC).

Gene Expression Analysis. Total RNA was isolated from mouse tissue andcells using TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μgof DNase-treated total RNA using SuperScript II reverse transcriptase(Invitrogen). mRNA levels were quantified by QPCR with SYBR Green(Invitrogen). Samples were run in technical triplicates, and relativemRNA levels were calculated by using the standard curve methodology andnormalized against 36B4 mRNA levels in the same samples.

Statistical analysis. All values are given as means±standard errors. Thetwo-tailed unpaired Student's t-test was used to assess the significanceof difference between two sets of data. Differences were considered tobe statistically significant when P<0.05.

Example 1 Identification of FGF-1 as a Direct Target of PPARγ

To identify nuclear hormone receptor (NHR) targets, we used a “PromoterOntology” screen, which encompasses a validated cDNA expression libraryincluding all 49 mouse NHRs combinatorially paired with a largecollection of pathway specific promoter-reporter libraries. The pairingfacilitates rapid evaluation of the transcriptional regulation of eachgenetic pathway by any NHR in a given context. Using thishigh-throughput promoter screen, we screened promoter constructs formembers of the FGF family for regulation by the NHRs, and identifiedFGF-1 as a direct target of PPARγ. More specifically, we identifiedstrong and specific transcriptional regulation of FGF-1 by PPARγ.

FGF-1A promoter characterization. The expression of the FGF-1 gene isdirected by at least three distinct promoters driving the untranslatedexons: 1A, 1B, and 1D, spaced up to 70 kilobase pairs apart (FIG. 1A)(Myers et al., 1993). Alternative splicing of these untranslated exonsto the three coding exons of the FGF-1 gene results in identical butdifferentially expressed FGF-1 polypeptides. In mice, FGF-1A shows thehighest expression in heart and kidney but is also expressed in adiposeand several other tissues (FIG. 1B). FGF-1B is the only variantexpressed in brain, and is also expressed in several other tissues (FIG.1C). FGF-1D is primarily expressed in liver (FIG. 1D).

The transcriptional regulation of FGF-1 by PPARγ was mediated throughbinding of PPARγ to a PPAR response element (PPRE) located in one of thealternative promoters of FGF-1, named FGF-1A (FIG. 2A). Inactivation ofthe PPRE in the FGF-1A promoter (located at −60 by relative to thetranscription start site (TSS)) by site directed mutagenesis resulted ina complete loss of response of the FGF-1A promoter to PPARγ (FIG. 2F,compare human vs. ΔPPRE).

The gene structure of FGF-1 is highly conserved in a wide range ofmammals (e.g., bovine, canine, horse, chimpanzee, orangutan, rat, mouse,and opossum). The PPRE in the FGF-1A promoter in these species alsoshowed strong conservation (FIG. 2D, E). To test the responsiveness ofthese PPREs to PPARγ, we changed the PPRE of the human FGF-1 promoter bysite directed mutagenesis into the PPRE sequence of species thatdisplayed sequence variation (rat, canine, horse, and opossum). PPARγactivation of the promoter was retained in all species except for themore distantly related canine and opossum (FIG. 2F). Together, thesefindings suggest a physiologically important function of regulation ofthe FGF-1A promoter by PPARγ, present in a wide range of mammals. Inaddition to a strong conservation of the PPRE in this promoter, severalother highly conserved elements were detected (e.g., SP1, HMTB, EVI1,and E-box).

The role of PPARγ in FGF-1 expression was confirmed in mature adiposecells. FIG. 21 shows the results of quantitative PCR, demonstrating thatPPARγ specifically binds the FGF1 promoter region. 36b4 is a negativecontrol locus that does not include PPARγ binding sites.

FGF-1 is regulated by PPARγ in vivo. Short term oral administration ofrosiglitazone (5 mg/kg for 3 days) or high-fat diet (two weeks)significantly increased the mRNA levels of FGF-1A in WAT (FIG. 3A, D).This increase was similar to that of the adipocyte protein aP2 (alsoknown as fatty acid binding protein 4, FABP4), which is the strongestknown PPARγ target in adipose tissue. On the other hand, overnightfasting resulted in an about two-fold decrease in FGF-1A mRNA levels.For comparison, levels of FGF-21 were highly induced in the liver byfasting and HFD (FIG. 3B, E) whereas no effects of rosiglitazone or HFDwere observed in WAT (FIG. 3C, F). Interestingly, rosiglitazone alsoreduced the expression of FGF-21 in fasted liver (FIG. 3B), which isalso observed in patients with type 2 diabetes (Li et al., 2009). Nochanges in expression by TZD, HFD, or fasting were observed for FGF-1Band FGF-1D in liver, and for FGF-1B in WAT. FGF-1A and FGF-1D were notdetected in liver and WAT, respectively. HFD treatment for 3 months inmice also resulted in increased protein levels of FGF-1 (FIG. 3G).

Example 2 FGF-1 Protects Against HFD-Induced Insulin Resistance

Next, we determined the consequences of loss of FGF-1 in vivo, usingFGF-1 knockout (KO) mice. FGF-1 KO mice have been studied in the contextof wound healing and cardiovascular changes. Neither these mice, norFGF-1/FGF2 double KO mice, displayed any significant phenotype undernormal feeding conditions (Miller et al., 2000). To study the role ofPPARγ-mediated regulation of FGF-1, FGF-1 KO and wild-type littermateswere fed a high fat diet (HFD). Although no difference in HFD-inducedweight gain was observed (FIG. 4A), FGF-1 KO mice had smaller WAT andlarger, steatotic livers, suggesting that FGF-1 KO mice fail to increasetheir adipose mass and alternatively mobilize fat into the liver (FIG.4B, C). At the same time, FGF-1 KO mice displayed increased fastinglevels of glucose and insulin and increased insulin resistance comparedto wild-type littermates as demonstrated by glucose- andinsulin-tolerance tests (GTT, ITT), respectively (Tables 1 and 2, FIG. 4D-F). No obvious abnormalities were observed in pancreas function asindicated by normal islet morphology, histology, and glucose-stimulatedinsulin secretion. The number of islets per pancreas, however, wasslightly increased (FIG. 4H).

TABLE 1 Metabolic parameters of male wild-type and FGF-1^(−/−) miceafter 3 months high fat diet feeding. wild-type FGF-1^(−/−) Insulin 0.32± 0.12 0.50 ± 0.34 ng/ml Glucose 103 ± 21  119 ± 24  mg/dl Leptin 6.3 ±2.0 6.7 ± 1.2 ng/ml Resistin 5.4 ± 1.4 5.6 ± 1.4 ng/ml IL-6 12.4 ± 3.6 13.9 ± 11.0 pg/ml TNFα 8.7 ± 1.2 8.1 ± 0.7 pg/ml MCP-1 48.0 ± 3.4   59.0± 3.4** pg/ml tPAI-1 0.58 ± 0.68 0.33 ± 0.33 ng/ml Body weight 39.9 ±2.8  40.9 ± 2.8  g

Results are expressed as mean serum concentrations after an overnightfast±SD, n=6; nd, *P<0.05.

TABLE 2 Metabolic parameters of male wild-type and FGF-1^(−/−) miceafter 5 months high fat diet feeding. wild-type FGF-1^(−/−) Insulin(fast) 2.7 ± 0.9  3.7 ± 0.4* ng/ml Glucose (fast) 159 ± 17  183 ± 29*mg/dl Adiponectin (fast) 12.9 ± 1.2  13.7 ± 1.8  μg/ml Total cholesterol(fed) 46.6 ± 12.3 44.6 ± 6.6  mg/dl Total cholesterol (fast) 49.2 ± 17.550.4 ± 3.1  mg/dl Free Fatty Acids (fed) 0.13 ± 0.02 0.11 ± 0.04 ng/mlFree Fatty Acids (fast) 0.20 ± 0.01 0.19 ± 0.02 ng/ml Triglycerides(fed) 16.88 ± 2.3  16.1 ± 1.9  mg/dl Triglycerides (fast) 11.2 ± 1.5 10.2 ± 0.9  mg/dl Body weight (BW)  46 ± 2.4  47 ± 1.2 g Liver weight1.9 ± 0.3  2.4 ± 0.3* g Liver % 4.1 ± 0.5  5.1 ± 0.5* % BW WAT weight1.9 ± 0.3  1.3 ± 0.1* g WAT % 4.4 ± 0.9  2.8 ± 0.2* % BW Kidney weight486 ± 30  463 ± 45  mg Heart weight 209 ± 12  196 ± 9  mg

Results are expressed as mean serum concentrations or weights±SD, n=5;nd, *P<0.05.

Example 3 AKT Signaling is Impaired in WAT of HFD-Fed FGF-1 KO Mice

FGFs signal through four cognate high-affinity tyrosine kinasereceptors, designated FGFR-1 to -4, leading to downstream activation ofmultiple signal transduction pathways, including the MAPK (ERK1/2) andPI3K/AKT pathways. These pathways regulate components of theinsulin/glucose signaling pathways including activation of glycogensynthase kinase-3 (GSK-3), which regulates glycogen synthesis inresponse to insulin, and translocation of the glucose transporter GLUT4(Cho et al., 2001). To investigate the integrity of these signalingpathways, we determined the expression of its critical components inWAT, BAT, liver, and muscle of HFD-fed FGF-1 KO and wild-type mice (FIG.5). Interestingly, we found that total levels of AKT (and to a lesserextent GSK3β) were reduced in WAT of HFD-treated FGF-1 KO mice comparedto WT mice. In contrast, levels of AKT were normal in liver, BAT, ormuscle, and levels of ERK1/2 were normal in all four tissues.

Example 4 FGF-1 Induces GLUT1 in Vitro

FGF-1 induces the expression of GLUT1 and acts synergistically withrosiglitazone in 3T3-L1 adipocytes. FGF-1 induces the expression ofGlucose Transporter 1 (Glut1) in mouse 3T3-L1 adipocytes after prolongedtreatment (FIG. 6), and it decreases fed blood glucose in ob/ob mice.The results indicate that FGF-1 can be used as a therapy for treatingdiabetes and obesity.

Example 5 FGF-1 has hypoglycemic effects in vivo

Eight-week-old male ob/ob mice were treated with recombinant mouse FGF-1(0.5 mg/kg/day, s.c. in 250 μl), rosiglitazone (TZD, 5 mg/kg/day, p.o.in 300 μl), or vehicle control (s.c. vehicle control 0.9% NaCl, 250μl/mouse; p.o. vehicle control 0.5% CMC, 300 μl/mouse). Blood glucosewas measured one hour after treatment at day 3 and day 6 using astandard protocol. (FIG. 7A).

Before treatment, all groups were severely hyperglycemic, as indicatedby blood glucose levels of about 400 mg/dl. At day three, bothFGF-1-treated and TZD-treated groups exhibited greatly reduced bloodglucose levels, about 200 mg/dl. At 6 days, blood glucose levels wereeven further reduced to around 130-140 mg/dl for both groups. After thesixth dose, blood glucose levels were monitored for another 72 hrs.During this period, both FGF-1- and TZD-treated cohorts maintainednormoglycemic levels (<140 mg/dl) for at least 48 hrs (FIG. 7B). At 72hrs after the sixth dose, a final dose was given, and 12 hrs later, atotal body composition analysis was performed by MRI followed bynecropsy.

The results show that FGF-1 is selectively induced in adipose tissue byhigh-fat diet (HFD) and TZD, and mice lacking FGF-1 develop HFD-inducedinsulin resistance (IR). At the molecular level, the IR of these micecan be explained by impaired AKT signaling in adipose. Administration ofFGF-1 to diabetic mice normalizes their glucose levels and improvestheir fat-lean ratio. Thus, FGF-1 acts as a powerful insulin sensitizerin adipose tissue and mediates insulin sensitizing actions of TZDs andPPARγ.

Example 6 FGF-1 Rapidly and Dramatically Reduces Glucose Levels in ob/obDiabetic Mice

In order to establish the acute effects of FGF-1 on blood glucoselevels, dose response curves (FIG. 8) and time courses aftersubcutaneous (FIG. 9) and intravenous (FIG. 10) administration wereperformed. The results show that FGF-1 causes dramatic dose-dependentreduction of glucose levels in ob/ob mice. Subcutaneous dosing iseffective within a matter of hours, and the significant reduction inglucose levels lasts at least 2 days (FIG. 9). FIG. 10 shows thatintravenous administration results in an even longer lasting effect onglucose levels, so that a dose of 0.2 mg/kg body weight resulted insignificantly reduced blood glucose for at least one week.

Example 7 Chronic Administration of FGF-1 Results in Normalized BloodGlucose Levels

To investigate the metabolic effects of chronic FGF-1 treatment in ob/obmice, eight weeks old male ob/ob mice were treated with vehicle orrecombinant mouse FGF-1 (0.5 mg/kg/3 days, s.c.) for a period of 36days. During this time, glucose levels, food intake, and bodycomposition were monitored. FIG. 11 shows that glucose levels arenormalized by the first time point tested (day 2) and remain stable forthe remainder of the test period.

Example 8 Administration of FGF-1 Results in Reduced Body Weight andPercent Body Fat

FIG. 12 shows that FGF-1 administration initially results in reducedfood intake of ob/ob mice. Food intake returns to normal within about 2weeks, but as shown in FIG. 13, body weight in FGF-1 treated ob/ob miceremains lower than in untreated ob/ob mice. The reduction in body weightshown in FIG. 13 indicates that FGF-1 can be used to produce rapid anddurable body weight reduction.

FIGS. 14 and 15 compare percent body fat and percent lean mass in FGF-1treated and untreated ob/ob mice. The results indicate that thereduction in body weight is largely due to reduced percentage body fat.The relative percentage of lean mass in FGF-1 treated mice issignificantly higher than in untreated mice (FIG. 15).

Example 9 FGF-1 Results in Improved Glucose Tolerance and ReducedInsulin Resistance

FIG. 16 shows the results of a glucose tolerance test carried out afterfour weeks of FGF-1 administration (0.5mg/kg/3 days, s.c.). FGF-1treated ob/ob mice cleared glucose more effectively than untreatedcontrols. FGF-1 treated mice also showed increased insulin sensitivity,as indicated by more rapid clearance of glucose in the ITT (FIG. 17).Serum lipid levels (triglycerides, free fatty acids, and cholesterol)were similar between the two groups (FIG. 18). These tests were carriedout as described above.

Example 10 FGF-1 Reduces Fatty Liver in ob/ob Mice

Analysis of liver tissue after the 36 day treatment period revealed thatthe livers of FGF-1 treated ob/ob mice were much healthier than theiruntreated counterparts. FIG. 19 shows H&E stained tissue from untreated(A) and treated (B) mice. The untreated liver displays significantsteatosis (fat deposit and damage), while the liver from FGF-1 treatedmice shows much less steatosis, and little if any inflammation.Moreover, liver glycogen levels were much higher in FGF-1 treated mice,which is indicative of proper glucose processing and insulin response(FIG. 20).

Example 11 Multiple Delivery Methods of FGF-1 are Effective for ReducingBlood Glucose

To determine if the effects of FGF-1 depend on the route ofadministration, we tested blood glucose levels of ob/ob mice in responseto 0.5 mg/kg body weight FGF-1 delivered s.c., i.p. and i.v. PBSinjections were used as controls. FIG. 22 shows that the acute effectsof FGF-1 are about the same for all three injection methods. We nextcompared i.v. and s.c. injections for duration of the glucosenormalizing effect. As shown in FIG. 23, FGF-1 administeredintravenously resulted in stable glucose levels for the duration of thetest, at least 60 hours. The data from FIG. 10 indicate that the effectsof intravenous injection are indeed much longer lasting (at least oneweek).

Example 12 FGF-1 is Effective for Normalizing Glucose in Other DiabeticModels

The ob/ob model is considered to represent a very severe diabeticdisease. In order to investigate the effect of FGF-1 on less severediabetic/metabolic disorder models, we tested blood glucose levels indb/db mice and diet induced obese mice. FIGS. 24 and 25 show thatsubcutaneous administration of 0.5 mg/kg FGF-1 was effective forreducing blood glucose levels in both systems. The data indicate thatFGF-1 can be used to normalize glucose levels and treat metabolicdisorders arising from different causes.

Example 13 Human Recombinant FGF-1 Effectively Reduces Glucose Levels inob/ob Mice

FIG. 26 shows that the same dose of hrFGF-1 administered s.c. caneffectively reduce glucose levels in ob/ob mice. As human recombinantFGF-1 is already being used in the clinic, the present methods of usingit to treat metabolic disorders offer a straightforward regulatory pathto treatment.

Example 14 Glucose Reducing Effects are Specific to FGF-1

As explained above, the FGF family of factors bind to members of theFGFR family of receptors with different specificities. FGF-1 bindspreferentially to FGFR1 and FGFR4, and can be internalized into a cellexpressing these receptors. To determine if other FGF proteins havesimilar metabolic effects as FGF-1, we tested blood glucose in ob/obmice treated with FGF-2, FGF-9, and FGF-10 (0.5 mg/kg s.c.). Thiscombination of FGF proteins binds to the spectrum of FGFRs. The resultsshown in FIG. 27 demonstrate that the particular receptor binding andsignaling properties of FGF-1 are required for the observed metaboliceffects.

1. A method for treating a diabetic, hyperglycemic, and/or insulinresistant individual who has a body mass index (BMI) of 25 or higher,comprising administering a functiional fragment of FGF-1 comprising atleast one variation at an amino acid residue position selected fromK112, K113, K118, R122, or K128 to the individual in an amount effectiveto reduce body fat in the individual and/or increase lean muscle mass inthe individuals.
 2. The method of claim 1, wherein the functionalfragment of FGF-1 compound is administered intravenously.
 3. The methodof claim 1, wherein the functional fragment of FGF-1 is administeredsubcutaneously.
 4. The method of claim 1, wherein the functionalfragment of FGF-1 is administered at a dose equivalent to 0.01-1 mgFGF-1 per kg body weight.
 5. The method of claim 1, wherein thefunctional fragment of FGF-1 is administered once per day or less. 6.The method of claim 1, wherein the functional fragment of FGF-1 isadministered in combination with an additional therapeutic compound. 7.The method of claim 6, wherein the additional therapeutic compound is analpha-glucosidase inhibitor, amylin agonist, dipeptidyl-peptidase 4(DPP-4) inhibitor, meglitinide, sulfonylurea, or a peroxisomeproliferator-activated receptor (PPAR)-gamma agonist.
 8. The method ofclaim 7, wherein the PPAR-gamma agonist is a thiazolidinedione (TZD),aleglitazar, farglitazar, muraglitazar, or tesaglitazar.
 9. The methodof claim 8, wherein the TZD is pioglitazone, rosiglitazone,rivoglitazone, or troglitazone.
 10. The method of claim 1, wherein thefunctional fragment of FGF-1 comprises at least 80% of human FGF-1. 11.The method of claim 10, wherein the functional fragment of FGF-1comprising at least 80% of human FGF-1 comprises at least 80% sequenceidentity to amino acids 1-140, amino acids 1-141, amino acids 14-135, oramino acids 13-135 of FGF1. 12.-13. (canceled)
 14. The method of claim10, wherein the functional fragment of FGF-1 comprising at least 80% ofhuman FGF-1 comprises at least 80% sequence identity to amino acids14-135 of FGF-1. 15.-16. (canceled)
 17. The method of claim 10, whereinthe functional fragment of FGF-1 comprising at least 80% of human FGF-1comprises at least 90% sequence identity to amino acids 1-141 of FGF-1.18. The method of claim 10, wherein the functional fragment of FGF-1comprising at least 80% of human FGF-1 comprises at least 95% sequenceidentity to amino acids 1-141 of FGF-1.
 19. The method of claim 10wherein the functional fragment of FGF-1 comprising at least 80% ofhuman FGF-1 comprises at least 98% sequence identity to amino acids1-141 of FGF-1.
 20. The method of claim 1, wherein the functionalfragment of FGF-1 is administered daily, twice daily, every other day,bi-weekly, weekly, or monthly. 21.-22. (canceled)
 23. The method ofclaim 1, wherein the individual has a BMI of greater than
 30. 24. Themethod of claim 1, wherein the individual has a BMI of 35 to
 40. 25. Themethod of claim 1, wherein the at least one variation is a conservativevariation.
 26. A method for treating a diabetic, hyperglycemic, and/orinsulin resistant individual who has a fatty liver disease, comprisingadministering a functional fragment of FGF-1 comprising at least onevariation at an amino acid residue position selected from K112, K113,K118, R122, or K128 to the individual in an amount effective to reduceliver steatosis.
 27. The method of claim 26, wherein the fatty liverdisease is nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liverdisease (NAFLD), simple fatty liver (steatosis), cirrhosis, or liverfibrosis.
 28. A method for treating an individual having type IIdiabetes or who is insulin resistant, comprising administering afunctional fragment of FGF-1 comprising at least one variation at anamino acid residue position selected from K112, K113, K118, R122, orK128 to the individual in an amount effective to reduce blood glucoselevels.